1. Field of the Invention
Cryptosporidium parvum is a protozoan parasite that has been implicated in numerous outbreaks of diarrheal disease in the human population. This invention relates to an isolated 41 kDa protein, CP41, specific for C. parvum; recombinant CP41(rCP41) proteins: a recombinant 36 kDa protein and a recombinant 28 kDa protein, both of which are specific for C. parvum; and the nucleic acid sequences which encode these proteins. The DNA which encodes CP41 and rCP41 can be used to specifically identify C. parvum oocytes through RT-PCR. The isolated and recombinant proteins can be used as reagents to detect antibodies in the serum of infected individuals, to make monoclonal antibodies that are specific for the native 41 kDa protein which specifically identifies C. parvum and thus distinguishes C. parvum from other Cryptosporidium species, to generate hyperimmune serum or colostrum for use in enhancing the anti-cryptosporidial response of young or immunocompromised individuals, and in vaccine development, to protect individuals from Cryptosporidium infection.
2. Description of the Relevant Art
Cryptosporidium is a protozoan that can cause acute, severe, self-limited disease in immunocompetent individuals and severe chronic diarrhea in immunocompromised individuals. The young and immunosuppressed are at particularly high risk. Worldwide, there is a much higher prevalence in children than in adults (Kehl et al 1995. J. Clin. Micro. 33 (2): 416-418. Although in most individuals the disease is self-limiting and protective immunity develops after a primary infection, cryptosporidiosis is a major cause of death in immunodeficient hosts such as persons afflicted with AIDS. Development normally takes place in the intestinal epithelium and the transmissible stage, the oocyst, is excreted in the feces. In immunocompromised patients, cryptosporidiosis is not necessarily self-limiting and sites other than the small intestine, such as the respiratory tract, stomach, liver, pancreas, gall bladder, appendix, colon, rectum, and conjunctiva of the eye, may be affected (Fayer et al. 1997. In Cryptosporidium and Cryptosporidiosis, R. Fayer, Ed., CRC Press, New York, N.Y. page 29). Cryptosporidiosis is also a major disease of dairy and beef calves in the United States. Although a number of species of Cryptosporidium have been described, only C. parvum causes disease in both humans and calves.
Over the last decade, this protozoan parasite has been implicated in numerous outbreaks of diarrheal disease in the human population. The largest outbreak reported to date was almost five years ago in Milwaukee, Wis. where greater than 400,000 people showed clinical signs of cryptosporidiosis. The source of the parasite was traced to contaminated drinking water supplied by a municipal water treatment utility. Such widespread occurrence of Cryptosporidium oocysts in raw and treated drinking water supplies throughout the USA has raised concern that low-level endemic waterborne Cryptosporidium infections may occur commonly.
Cryptosporidium is transmitted through animal contact, person-to-person contact, and contaminated food and water. The C. parvum infection is initiated by the ingestion of oocysts, the excystation of oocysts with release of sporozoites and the invasion of gut epithelial cells by sporozoites. Thereafter, the intracellular forms mature and release new daughter merozoites which reinvade the gut epithelial cells. C. parvum also has a sexual cycle. The sexual cycle of C. parvum also occurs in the gut and results in the production of sporulated oocysts, some of which may excyst before being shed. In persistent infection of an immunocompromised host, both the merozoite and the endogenously produced sporozoite may contribute to the ongoing invasion by C. parvum. Cryptosporidium spp. are resistant to standard disinfection processes and remain infectious for long periods of time in the environment at a wide range of temperatures. This resistance is imparted by the hard outer covering of the oocyst wall that surrounds the infectious stage of the parasite, ie., sporozoites.
The detection of Cryptosporidium parvum oocysts in environmental samples usually relies on one of three different techniques--vital dye staining (e.g., Modified Ziehl-Neelsen acid fast staining), direct or indirect immunofluorescence staining (IFA), or enzyme immunoassay (EIA) using Cryptosporidium-reactive antibodies. Differences in the relative sensitivities of these assays have been noted (Garcia et al. 1997. J. Clin. Micro. 35 (6): 1526-1529; Graczyk et al. 1996. Am. J. Trop. Med. Hyg. 54(3): 274-279; Ignatius et al. 1997. Euro. J. Clin. Micro. Inf. Dis. 16: 732-736; and Kehl et al. 1995. J. Clin. Microbiol. 33: 416-418). The majority of immunocompetent patients, when initially symptomatic, have large numbers of oocysts present in their stools and their condition can be confirmed with a number of procedures; however, as the acute infection resolves, the patient becomes asymptomatic and the number of oocysts dramatically decreases (Garcia et al. 1997, supra). Low numbers of oocysts makes identification of C. parvum as the causative agent difficult. The high sensitivity of anti-Cryptosporidium monoclonal antibodies (mAbs) most certainly aids detection of Cryptosporidium in fecal or environmental samples; however, their use does not ensure the specific detection of C. parvum, the only species that represents potential public health threats. Cryptosporidium oocysts shed by a variety of captive and wild homoiothermal and poikilothermal animals contaminate the surface water and water supply. In the absence of C. parvum-specific mAbs, such oocysts can be misinterpreted as C. parvum oocysts potentially pathogenic for humans based on their identification as Cryptosporidium oocysts by crossreactive antibodies, i.e., antibodies that react with more Cryptosporidium species than C. parvum (Graczyk et al. 1996. Am. J. Trop. Med. Hyg. 54(3): 274-279). Similarly, diarrhea in patients may be inaccurately diagnosed as resulting from C. parvum under circumstances where an organism other than C. parvum is the causative agent and the patient carried Cryptosporidium oocysts (not C. parvum) from contacts not related to the diarrheal disease, i.e., environmental contacts. This problem is of particular concern for water treatment utilities that must monitor the efficiency of filtration processes and the contamination level of treated water destined for human consumption. None of the available immunoassays can differentiate C. parvum from other species of Cryptosporidium that are not infectious for mammals. The inability to sensitively detect and differentiate Cryptosporidium at the level of species or subspecies (strain) is a recognized constraint on our understanding of the natural history, epidemiology, and zoonotic potential of Cryptosporidium isolates and therefore makes the assessment of the public health risk posed by oocyst contamination of water or foods difficult (M. J. Arrowood. 1997. In Cryptosporidium and Cryptosporidiosis, R. Fayer, Ed., CRC Press, New York, N.Y. page 56).
Confirmatory diagnosis of cryptosporidiosis in patients is often carried out by assaying sera for recognition of specific Cryptosporidium antigens (Frost et al. 1998. Epidemiol. Infect. 121: 205-211). Several low molecular weight C. parvum oocyst antigens, such as 15 kDa, 17 kDa, and 23 kDa proteins, appear to be useful for identifying the presence of Cryptosporidium. The immunogenicity of 15, 17, and 23 kDa antigens and somewhat higher M.sub.r antigens (e.g., 32, 47 kDa) has been observed in other mammalian species infected or immunized with C. parvum oocysts (Lorenzo et al. 1995. Vet. Parasitol. 60: 17-25; McDonald et al. 1992. Parasite Immunol. 14: 227-232; Nina et al. 1992. Infect. Immun. 60: 1509-1513; Peeters et al. 1992. Infect. Immun. 60: 2309-2316; Perryman et al. 1996. Mol. Biochem. Parisitol 80:137-147; Reperant et al. 25 1994. Vet. Parasitol. 55: 1-13). However, laboratory studies have shown these immunodominant antigens and other oocyst/sporozoite proteins to be present in other Cryptosporidium species (Nina et al. 1992, supra; Tilley et al. 1990. Infect. Immun. 58: 2966-2971); therefore, their presence is not indicative of C. parvum infection. This cross-reactivity of immunodominant antigens may explain why commercial antibody-based tests cannot differentiate C. parvum from species of Cryptosporidium that are not infectious for humans.
No antibiotics or antiprotozoal drugs licensed for animal use have been approved for prophylaxis or therapy of cryptosporidiosis (Fayer et al. 1997. In Cryptosporidium and Cryptosporidiosis, R. Fayer, Ed., CRC Press, New York, N.Y., pages 20 and 30-31). Several researchers have shown, however, that in calves, mice and humans, administration of hyperimmune bovine colostrum, prepared by immunizing cows with extracts of C. parvum oocysts, can effectively confer passive immunity against cryptosporidiosis (Fayer et al. 1989. J. Parasitol. 75(1):151-153; Fayer et al. 1989. J. Parasitol. 75(3):393-397; Fayer et al. 1990. Infect. Immun. 58(9):2962-2965; Nord et al. 1990. AIDS 4:581-584; Tzipori et al. 1986. Br. J. Med. 293:1276-1277; Tzipori et al. 1987. Lancet 2:244-245; and Ungar et al. 1990. Gastroenterology 98:486-489). Duodenal infusions of hyperimmune bovine colostrum have been reported to ameliorate C. parvum infection in AIDS or other immunocompromised patients. Hyperimmune bovine colostrum prepared against oocysts contains neutralizing antibodies that recognize epitopes expressed by all life-cycle stages of Cryptosporidium.
Monoclonal antibodies and immune sera that bind to C. parvum sporozoites can neutralize the parasite and either prevent or lessen the severity of infection in animals. The characteristics of many mAbs which specifically react with Cryptosporidium have recently been reviewed; many are neutralizing (Riggs. 1997. In Cryptosporidium and Cryptosporidiosis, R. Fayer, Ed., CRC Press, New York, N.Y., Chapter 6). In some instances, the epitope recognized by the monoclonal antibodies has been found in both sporozoites and merozoites. These monoclonal antibodies have been shown to prevent or attenuate infection in studies using animals challenged with C. parvum. However, none of these mAb specifically bind exclusively to C. parvum. Thus, they cannot be used to specifically identify the presence of C. parvum in environmental or patient samples.
Riggs reviews other mAbs reactive with surface membrane antigens and/or apical complex organelles of sporozoites and merozoites; however, no neutralization data are reported (Riggs. 1997, supra). Based on the proteins bound by these mAb and the species involved, these mAb do not selectively identify C. parvum-specific proteins.
Although protection against C. parvum may be achieved by this type of immunotherapy, the development of resistance to cryptosporidiosis is dependent upon T lymphocytes and secreted lymphokines, in particular, gamma-interferon (Gardner. 1991. Am. J. Trop. Med. Hyg. 44(1):49-62; Mead et al. 1991. J. Infect. Dis. 163:1297-1304; McDonald et al. 1992. Infect. Immun. 60 (8):3325-3331; and Ungar et al. 1991. J. Immunol. 147 (3):1014-1022). The humoral response leading to production of protective antibodies specific for C. parvum may be dependent upon T cell signaling, but in persons with severe immunodeficiency T cell-mediated immunity is dysfunctional. Passive administration of hyperimmune serum or colostrum that is inhibitory for cryptosporidial parasites may be the only viable alternative for preventing or treating infection in such individuals.
Thus, there is a need for agents useful for the immunotherapy of cryptosporidiosis in both uncompromised and immunocompromised subjects, e.g. AIDS patients, which would prevent or limit the disease. There is also a need for an agent useful for detection of C. parvum in environmental samples and for the detection of ongoing C. parvum invasion, particularly in its early stages.